Characteristics of Glutathione Sepharose and HiTrap^® Benzamidine FF (High Sub) Media and Columns

Glutathione Sepharose High Performance is recommended for high-resolution purification of GST-tagged proteins, providing sharp peaks and concentrated eluent. Glutathione Sepharose 4 Fast Flow is excellent for scaling up. Glutathione Sepharose 4B has high capacity and is recommended for packing small columns and other formats including batch purifications.

Table A5.1 summarizes key characteristics of these three Glutathione Sepharose media, and Tables A5.2 to A5.6 summarize the characteristics of these media prepacked in columns and in 96-well filter plates. Table A5.7 summarizes key characteristics of HiTrap Benzamidine Fast Flow (high sub).

¹ The binding of GST-tagged proteins depends on size, conformation, and concentration of the protein in the sample loaded. Binding of GST to glutathione is also flow dependent, and lower flow rates often increase the binding capacity. This is important during sample loading. Protein characteristics, pH, temperature, and the media used may also affect the binding capacity.

² When using water at room temperature.

¹ According to American National Standards Institute (ANSI) and Society for Biomolecular Screening (SBS).

1-2004, 3-2004, and 4-2004 standards.

² The amount of eluted target proteins/well does not differ more than +/- 10% from the average amount/well for the entire filter plate.

¹ The binding of GST-tagged proteins depends on size, conformation, and concentration of the protein in the sample loaded. Binding of GST to glutathione is also flow dependent, and lower flow rates often increase the binding capacity. This is important during sample loading. Protein characteristics, pH, temperature, and the media used may also affect the binding capacity.

² Dynamic binding capacity conditions (60% breakthrough):

Sample: 1 mg/ml pure GST-tagged protein in binding buffer
Column volume: 0.4 ml
Flow rate: 0.2 ml/min (60 cm/h)
Binding buffer: 10 mM sodium phosphate, 140 mM NaCl, 2.7 mM KCl, pH 7.4
Elution buffer: 50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0

³ When using water at room temperature.

¹ The binding of GST-tagged proteins depends on size, conformation, and concentration of the protein in the sample loaded. Binding of GST to glutathione is also flow dependent, and lower flow rates often increase the binding capacity. This is important during sample loading. Protein characteristics, pH, temperature, and the media used may also affect the binding capacity.

² Dynamic binding capacity conditions (60% breakthrough):

Sample: 1 mg/ml pure GST-tagged protein in binding buffer
Column volume: 0.4 ml
Flow rate: 0.2 ml/min (60 cm/h)
Binding buffer: 10 mM sodium phosphate, 140 mM NaCl, 2.7 mM KCl, pH 7.4
Elution buffer: 50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0

³ When using water at room temperature.

Note: It is not recommended to autoclave the columns.

¹ Binding capacity is protein dependent. The binding of GST-tagged proteins depends on size, conformation, and concentration of the protein in the sample. Binding of GST to glutathione is also flow dependent, and lower flow rates often increase the binding capacity. This is important during sample loading. Protein characteristics, pH, and temperature may also affect the binding capacity.

² Exposing the sample to 6 M Gua-HCl will denature the GST tag. It is therefore important to remove all Gua-HCl before use.

¹ Binding capacity is protein dependent. The binding of GST-tagged proteins depends on size, conformation, and concentration of the protein in the sample loaded. Binding of GST to glutathione is also flow dependent, and lower flow rates often increase the binding capacity. This is important during sample loading. Protein characteristics, pH, and temperature may also affect the binding capacity.

² Exposing the sample to 6 M Gua-HCl will denature the GST tag. It is therefore important to remove all Gua-HCl before use.

¹ The ranges given are estimates based on our knowledge and experience. Please note the following: pH stability, short term refers to the pH interval for regeneration, cleaning-in-place, and sanitization procedures. pH stability, long term refers to the pH interval where the medium is stable over a long period of time without adverse effects on its chromatographic performance.